The adaptive immune response starts when CD4+ T cells recognize antigenic peptides presented by class II molecules of the Major Histocompatibility Complex (MHCII). In general, this response focuses on very few representative peptides from each possible pathogenic agent, referred to as immunodominant epitopes. Though immunodominance has been observed for several years, the underlying mechanisms are still unclear. Two outstanding features of MHCII molecules complicate our ability to understand immunodominance: their extensive polymorphism and the ability of each allele to bind large panoply of peptides. A third confounding factor is that epitope selection usually takes place in the presence of HLA-DM (DM), of which activity, not fully elucidated, results in the generation of an antigenic repertoire skewed toward kinetically and energetically stable peptide-MHCII complexes (pMHCII). Our long-term goal is to formally describe the stages of the epitope selection and recognition process. A better grasp on the mechanisms involved in the generation of dominant epitopes and T cell responses to them is important in areas where MHCII plays a significant role, such as vaccination and transplantation. This proposal addresses immunodominance at the level of the peptide and the MHC. We have been investigating the pMHCII interaction with a different approach than the typical kinetic analysis, by looking at the thermodynamic aspects of complex formation. We have shown for the human MHCII HLA-DR1 (DR1) that different peptides can bind with comparable affinities adopting different thermodynamic mechanisms. In addition, we have shown that complexes, of which formation requires smaller entropic penalty, feature greater conformational flexibility and are more susceptible to DM action. Our observations lead us hypothesize that a correlation exists between: 1) peptide binding thermodynamics, 2) pMHCII structure and conformational flexibility and 3) DM-susceptibility. Moreover, we hypothesize that binding and DM-susceptibility may alternatively control or being controlled by the availability of candidate epitopes as defined by endosomal proteolysis, and together these three processes determine the selection of immunodominant epitopes. To test these hypotheses, in Aim 1 we will consider a library of peptides derived from HA H1N1 for which DR1- restricted dominance hierarchy has been established, and we will analyze binding and kinetic properties of these peptides in complex with DR1. Moreover, for a subset of sequences, we will probe the correlation between thermodynamics of binding, structural flexibility of the respective complexes and DM-susceptibility. In Aim 2 we will incubate, together or in sequence, the whole H1 protein with cathepsins, DR1 and DM, we will probe the kinetics of reaction in 2D gels and we will use mass spectrometry (MS) to analyze sequences eluted from DR1 at the end of each incubation. We will compare the H1 epitope hierarchy with the MS-identified sequences to determine the relative role of proteolysis and DM-susceptibility in the generation of dominance.